Icaritin Sensitizes Hepatocellular Carcinoma to PD-L1 Therapy by NQO1-Dependent Ferroptosis Induction

Hepatocellular carcinoma (HCC) remains challenging with limited immunotherapy response. Despite its clinical promise in advanced HCC, the mechanisms of icaritin, especially concerning ferroptosis induction and immune modulation, remain elusive. This study aims to determine if the antitumor effect of icaritin involves the induction of ferroptosis via NAD(P)H quinone oxidoreductase 1 (NQO1) and if it can augment the efficacy of programmed cell death 1 ligand 1 (PD-L1) therapy by potentiating natural killer (NK) cell activity. Using human HCC cell lines (Huh7, Hep3B, PLC/PRF/5, SNU-449, and MHCC97-H) and two synergistic mouse models (Hepa1-6 and SgPten/c-Met), we examined icaritin's inhibition of tumor growth and induction of ferroptosis via the NQO1 pathway, monitoring key markers (reactive oxygen species [ROS], glutathione peroxidase 4 [GPX4], ferritin heavy chain 1 [FTH1]). The NQO1 inhibitor dicoumarol was employed to validate the pathway. Tumor microenvironment (TME) remodeling was assessed through cancer-associated fibroblasts (CAFs) markers and immune cell profiling, focusing on NK cell infiltration. Combination therapy with anti-PD-L1 was tested in vivo. Icaritin significantly inhibited HCC growth in vitro and in vivo. Its antitumor effect was mediated by NQO1-mediated ferroptosis, via elevated ROS, diminished mitochondrial membrane potential, and downregulated GPX4 and FTH1. Analysis of The Cancer Genome Atlas (TCGA) data revealed that NQO1 is overexpressed in human HCC tissues. Icaritin enhanced NK cell infiltration while reducing CAF abundance and suppressing recombinant focal adhesion kinase (FAK) and discoidin domain receptor 1 (DDR1) signaling. Notably, icaritin synergized with anti-PD-L1 therapy to enhance tumor suppression without increasing toxicity, correlating with potentiated NK cell immunity. Our findings demonstrate that icaritin triggered NQO1-mediated ferroptosis and remodeled TME to enhance NK cell recruitment and PD-L1 therapy efficacy. This provides rationale for evaluating icaritin-based combination immunotherapy in HCC through dual action on ferroptosis and NK cell activation.